Life in an oxygen rich environment presents a number of challenge. Due to the spin forbidden and, hence, kinetically sluggish reaction of triplet dioxygen with organic metabolites, enzymes generally catalyze single electron transfer steps to generate dioxygen-derived radical species as intermediates. While overcoming kinetic barriers, single electron reaction paths introduce the potential for free radical damage to macromolecular components within the cell. This project is aimed at understanding the strategies that enzyme have evolved to catalyze the production of reduced oxygen intermediates while minimizing/preventing oxidative damage. A set of protocols have been developed that focus on the second order rate constant for reaction of dioxygen with enzyme and allow a detailed characterization of all steps from dioxygen binding to protein up through the first irreversible step of catalysis. Some of the questions that can be addressed using these methods are: What is the nature of rate determining steps in the reaction of dioxygen with proteins? Do chemical intermediates accumulate and, if so, what is their identity? To what extent, if any, are the steps that involve electron and proton transfer coupled? What is the role of active site residues within a protein in bringing about catalysis of dioxygen activation? and Are there simple rules or patterns that can be expected to emerge from experimental probes of the above questions? Studies are planned with a wide range of enzyme systems that vary with regard to the nature of their active sites. These enzyme systems include those that function solely in the presence of metal centers (dopamine beta- monooxygenase, peptide amidating enzyme, lipoxygenase and methane monooxygenase), enzymes that use organic cofactors alone (glucose oxidase), enzymes that contain both metal centers and organic cofactors at their active sites (galactose oxidase and cytochrome P-450) and enzymes that undergo reaction with dioxygen in the absence of either a metal center or organic cofactor (rubisco).